1. CRP Structure and Activity
C-reactive protein was first described by Tillett and Francis [J. Exp. Med., 52:561-71 (1930)] who observed that sera from acutely ill patients precipitated with the C-polysaccharide of the cell wall of Streptococcus pneumonia. Other investigators subsequently identified the reactive serum factor as protein, hence the designation "C-reactive protein" or "CRP." Kilpatrick et al., Immunol. Res., 10:43-53 (1991), provides a recent review of CRP.
CRP is a pentameric molecule which consists of five identical subunits [Osmand et al., Proc. Natl. Acad. Sciences, U.S.A., 74: 739-743 (1977)]. This pentameric form of CRP is sometimes referred to as "native CRP."
The gene sequence for human CRP has been cloned [Lei et al., J. Biol. Chem., 260:13377-13383 (1985)]. In addition, the primary sequences for rabbit CRP [Wang et al., J. Biol. Chem., 257:13610-13615 (1982)] and murine CRP have been reported [Whitehead et al., Biochem. J., 266:283-290 (1990)], and is under investigation for rat, dog, horse, goat, and sheep. Clinical and laboratory observations have determined that the acute phase response, classically defined by the well-defined changes of the blood [Pepys et al., Advances in Immunology, 34:141-212 (1983)], develops during various states of disease and injury including malignant neoplasia, ischemic necrosis, and bacterial, viral, or fungal parasitic infections. Measurement of serum acute phase reactants such as CRP have been utilized in clinical tests for diagnosis and clinical management of patients with various conditions, including systemic lupus erythematosus (SLE) [Bravo et al., J. Rheumatology, 8:291-294 (1981)], rheumatoid arthritis [Dixon et al., Scand. J, Rheumatology, 13:39-44 (1984)], graft versus host disease [Walker et al., J. Clin. Path., 37.:1022-1026 (1984)], as well as many other diseases.
2. Modified-CRP Structure and Activity
In about 1983, another form of CRP was discovered which is referred to as "modified C-reactive protein" or "mCRP." mCRP has significantly different charge, size, solubility and antigenicity characteristics as compared to native CRP [Potempa et al., Mol. Immunol., 20:1165-75 (1983)]. mCRP also differs from native CRP in its binding characteristics. For instance, mCRP does not bind phosphorylcholine [Id,; Chudwin et al., J. Allergy Clin. Immunol., 77:216a (1986)].
The distinctive antigenicity of mCRP has been referred to as "neo-CRP." Neo-CRP antigenicity is known to be expressed on:
1) CRP treated with acid, urea or heat under certain conditions; PA1 2) the primary translation product of DNA coding for human and rabbit CRP; and PA1 3) CRP immobilized on plastic surfaces [Potempa et al., Mol. Immunol., 20:1165-75 (1983); Mantzouranis et al., Ped. Res., 18:260a (1984); Samols et al., Biochem. J., 227:759-65 (1985); Potempa et al., Mol. Immunol., 24:531-541 (1987)]. A molecule reactive with polyclonal antibody specific for neo-CRP has been identified on the surface of 10-25% of peripheral blood lymphocytes (predominantly NK and B cells), 80% of monocytes, and 60% of neutrophils, and as well as at sites of tissue injury [Potempa et al., FASEB J.,2:731a (1988); Bray et al., Clin. Immunol. Newsletter, 8:137-140 (1987); Rees et al., Fed, Proc., 45:263a (1986)].
Furthermore, mCRP differs from native CRP in its biological activity. It has been reported that mCRP can influence the development of monocyte cytotoxicity, improve the accessory cell function of monocytes, potentiate aggregated IgG-induced phagocytic cell oxidative metabolism, and increase the production of interleukin-1, prostaglandin E and lipoxygenase products by monocytes [Potempa et al., Protides Biol. Fluids, 34:287-290 (1987); Potempa et al., Inflammation, 12:391-405 (1988); Chu et al., Proc. Amer. Acad. Cancer Res., 28:344a (1987); Potempa et al., Proc. Amer, Acad. Cancer Res., 28:344a (1987); Zeller et al., Fed, Proc., 46:1033a (1987); Chu et al., Proc. Amer. Acad. Cancer Res., 29:371a (1988)].
In vivo experiments with mCRP were performed to determine if mCRP was capable of providing a protective effect against lethal doses of Streptococcal pneumonia [Chudwin et al., J. Allergy Clin. Immunol., 77:216a (1986)]. These studies demonstrated that intravenous administration of mCRP not only protected the animals from lethal S. pneumonia doses but that mCRP efficacy was 3 to 4 fold greater than native CRP.
3. Hematopoietic Cell Production and Activity
Pluripotent stem cells in the bone marrow of mammals have the potential to give rise to different types of blood cells which circulate in the peripheral blood. The pluripotent stem cells differentiate into various cell lineages through multiple maturational stages, thereby giving rise to committed blood cell types.
One cell lineage differentiated in the bone marrow is the megakaryocytic lineage. The earliest recognizable member of the megakaryocytic lineage is the megakaryoblast. A morphologic classification commonly applied to the megakaryocyte lineage refers to the megakaryoblast as the earliest cell form, the promegakaryocyte or basophilic megakaryocyte as the intermediate cell form, and the mature megakaryocyte as the final cell form. The mature megakaryocyte then forms and extends filaments of cytoplasm (also called pseudopods) that detach and fragment into individual thrombocytes or platelets. The process of thrombocyte formation, starting from the earliest blast cell stage, typically takes about three days.
Thrombocytes generally circulate in the peripheral blood and play an important role in the body's response to injury or trauma. For example, thrombocytes can become activated and aggregate at the site of injury or trauma. Thrombocytes are also secretory cells, containing various granules which are the secretory organelles of the cell. Such granules include alpha granules, dense granules, and lysosomal granules [Harrison's Principles of Internal Medicine, 9th Ed., McGraw-Hill, New York, 1980].
Alpha granules are typically the first granules released from thrombocytes. The alpha granules contain several proteins, including platelet factor 4, beta-thromboglobulin, and fibrinogen. The dense granules are usually released after the alpha granules. The dense granules contain calcium, ADP, serotonin, and catecholamines. The lysosomal granules are usually released last and contain enzymes such as phosphatase, beta-glucuronidase, and cathespin. These proteins and enzymes are released from the thrombocyte granules in response to certain stimuli and assist in potentiating thrombus, or blood clot, formation.
The effects of native CRP and altered forms of CRP on the activation, aggregation, and secretory function of circulating thrombocytes has been the subject of several reports. Fiedel et al., Immunology, 45:439-447 (1982), describe that thermally-aggregated CRP induced isolated platelets to aggregate and secrete in in vitro culture. Fiedel et al. also discuss the ability of aggregated CRP to initiate platelet responsiveness and enhance platelet activation in plasma stimulated by various platelet agonists [See also, Fiedel et al., Clin. Exp. Immunol., 50:215-22 (1982)].
Miyazawa et al., J. Immunol., 141:570-74 (1988), report that FA-CRP (defined as human CRP treated with an Fe.sup.2+ -ascorbate) in combination with suboptimal doses of platelet-activating factor and other stimulator agents activated platelets in vitro. The authors also observed that the FA-CRP did not show activity toward rabbit platelets, and therefore, concluded that the activity was species specific.
Fiedel, Blood, 65:264-69 (1985), disclose that aggregated human CRP in combination with suboptimal concentrations of ADP in platelet-rich plasma induced platelets to aggregate, secrete dense and alpha-granule constituents, and generate thromboxane A.sub.2.
Potempa et al., Inflammation, 12:391-405 (1988), disclose that mCRP is capable of activating platelets, PMNL, and monocytes in vitro. The authors also report that in certain culture conditions, mCRP activated platelets to aggregate.
Other investigations have focused on examining the processes by which thrombocytes are made and the factors which influence those processes. The production of thrombocytes has typically been viewed as a process involving two different stages. The first stage is directed to proliferation or differentiation of megakaryocytes. The second stage is directed to maturation or release of the megakaryocytes into thrombocytes. A review of thrombocytopoiesis and megakaryocytopoiesis is provided in The Platelet, an International Academy of Pathology Monograph, published by The Williams & Wilkins Company, 1971.
It is generally recognized that different factors are needed for each stage of the production of thrombocytes [See, e.g., Murphy, Hematol. Oncol. Clin. North Am., 3:465-478 (1989)]. A first factor is an inducer of the proliferation or clonal growth of megakaryocyte progenitors. This factor is sometimes referred to as Megakaryocyte Colony Stimulating Factor (Meg-CSF). A second factor is a promoter of maturation of megakaryocytes and formation and release of platelets. This factor is sometimes referred to as a Megakaryocyte-Potentiator (Meg-POT) factor. Factors which exhibit Megakaryocyte-potentiator activity typically have the ability to promote megakaryocyte colony formation in the presence of Meg-CSF, to stimulate megakaryocyte polyploidization, and induce the maturation of megakaryocytes.
Thrombocytopoiesis and megakaryocytopoiesis may be adversely affected by different diseases or pathological conditions. Cell production may also be adversely affected by radiation, drugs, or surgery. While therapies such as surgery, chemotherapy, and radiation have improved, they are nonetheless often accompanied by damage to bone marrow and/or other blood cell-producing tissues.
In diagnosing such conditions or monitoring the effects of certain therapies, platelet counts in the peripheral blood are typically measured. A decrease in the number of platelets in the blood can occur in certain medical disorders [Marchasin et al., California Medicine, 101:95-100 (1964)]. Thrombocytopenia, a medical condition characterized by a low platelet count in the blood, can result from impaired production of platelets by the bone marrow, platelet sequestration in the spleen, or increased destruction of platelets in the peripheral circulation. Further, for patients receiving large volumes of rapidly administered platelet-poor blood products, thrombocytopenia can develop due to dilution of the blood.
In addition to measuring platelet numbers, platelet volume, also referred to as mean platelet volume ("MPV"), can be measured. An increase in MPV has been associated with increased megakaryocyte size in response to thrombopoietic stress [Thompson et al., Blood, 72:1 (1988)]. It has also been observed that platelet count and MPV are inversely related, i.e., patients with low platelet counts tend to have larger MPV and patients with high platelet counts tend to have smaller MPV [Id.]. This inverse relationship has been interpreted as one mechanism by which the body maintains a relatively stable platelet mass. The "platelet mass" is determined by multiplying the platelet number by the mean platelet volume.
Thrombocytopenia and other conditions characterized by abnormal platelet numbers or volume have been treated in various ways. One method employed to treat thrombocytopenia is platelet transfusion. Platelet transfusions can be effective in some circumstances, but are undesirable because of the costs and risk of infections.
Certain cytokines, humoral factors, and chemical compounds have been identified as inducing thrombocyte production and megakaryocyte growth [See, for example, U.S. Pat. No. 5,126,325 (human B cell differentiation factor); U.S. Pat. No. 5,032,396 (interleukin-7); U.S. Pat. No. 5,250,732 (ketamine analogues); U.S. Pat. No. 5,260,417 (megakaryocyte growth promoting factor, "MGPA")]. In vitro, interleukin-3 (IL-3), interleukin 6 (IL-6) interleukin-11 (IL-11) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to increase the number and size of megakaryocyte colonies [Williams, Immunol. Ser., 49:215-229 (1990); Hoffman et al., Yale J. Biol. Med., 63:411-418 (1990); Yonemura et al., Exp. Hematol, 20:1011 (1992); Teramura et al., Blood, 79:327 (1992); Carrington et al., Blood, 77:34 (1991)]. IL-3 is believed to principally affect the differentiation (earliest) phase of the thrombopoiesis process [Moore et al., Blood, 78:1 (1991); Sonoda et al., PNAS USA, 85:4360 (1988)]. In contrast, IL-11 has been reported to influence thrombopoiesis principally at the maturation (later) phase [Teramura et al., Blood, 79:327 (1992); Yonemura et al., Exp. Hematol., 20:1011 (1992)].
Several investigators have reported that in vivo administration of interleukin-6 increased thrombocyte counts in the peripheral blood of primates [Asano et al., Blood, 75:1602 (1990)]and mice [Podja et al., Exp. Hematol., 18:1034 (1990); Ishibashi et al., Blood, 74:1241 (1989)]. Carter et al. reported that administration of exogenous thrombopoietin decreased the severity and duration of radiation-induced thrombocytopenia in mice [Radiation Research, 132:74-81 (1992)].